Production of biological macromolecules, particularly proteins, often involves purity-enhancing steps based on physical and physicochemical properties. Difficulties encountered in such process steps include, but are not limited to, determining conditions which enable separation of soluble and insoluble molecules, relatively low recovery of the desired molecule after a treatment step, loss of biological activity in the course of the process, and sensitivity of the protein to process step conditions such as pH.
Surfactants have been utilized in the processing of biological macromolecules. Cationic surfactants are a recognized subclass of surfactants, and include amphipathic ammonium compounds. Amphipathic ammonium compounds comprise quaternary ammonium compounds of the general formula QN+ and paraffin chain primary ammonium compounds of the general formula RNH3+. Both types of amphipathic ammonium compounds include long-chain ammonium surfactants that have a long aliphatic chain of preferably at least six carbon atoms (Scott (1960) Methods Biochem. Anal. 8:145-197, incorporated herein by reference in its entirety). The long-chain quaternary ammonium surfactants are known to interact with biological macromolecules. The long-chain quaternary ammonium compounds have at least one substituent at the nitrogen which consists of a linear alkyl chain with 6-20 carbon atoms. The best known representatives of this class are the benzalkonium salts (chlorides and bromides), hexadecylpyridinium chloride dequalinium acetate, cetyldimethylammonium bromide (CTAB) and hexadecylpyridinium chloride (CPCl), and benzethonium chloride. Quaternary ammonium surfactants include salts such as cetyl pyridinium salts, e.g. cetyl pyridinium chloride (CPC), stearamide-methylpyridinium salts, lauryl pyridinium salts, cetyl quinolynium salts, lauryl aminopropionic acid methyl ester salts, lauryl amino propionic acid metal salts, lauryl dimethyl betaine stearyl dimethyl betaine, lauryl dihydroxyethyl betaine and benzethonium salts. Alkyl pyridinium salts comprise stearyl-trimethyl ammonium salts, alkyl-dimethylbenzyl-ammonium chloride, and dichloro-benzyldimethyl-alkylammonium chloride.
Known uses of cationic surfactants for purifying biological macromolecules include 1) solubilization of aggregates, including protein aggregates; 2) elution of chromatographic column-bound biological macromolecules; and 3) precipitation of polyanions such as hyaluronic acid (HA), nucleic acids, and heparin (and molecules which co-precipitate with polyanions).
Cationic surfactants have been used for solubilizing protein aggregates. Otta and Bertini ((1975) Acta Physiol. Latinoam. 25:451-457, incorporated herein by reference in its entirety) demonstrated that active uricase could be solubilized from rodent liver peroxizomes with the quaternary ammonium surfactant, Hyamine 2389. It is found that increase of the ammonium surfactant concentration resulted in increase of dissolution of both uricase (based on enzymatic activity) and total protein such that there is no increase in the relative amount of uricase protein with respect to the amount of total protein. In other words, there was no selective solubilization of the uricase protein with respect to the total protein, and the uricase protein did not constitute a higher percentage of the total protein upon solubilization with the cationic surfactant. Thus, in this process, uricase purity with respect to the total protein content is apparently not enhanced as a result of quaternary ammonium surfactant solubilization.
In another study, Truscoe ((1967) Enzymologia 33:1 19-32, incorporated herein by reference in its entirety) examined a panel of cationic, anionic, and neutral detergents for their extraction efficacy of urate oxidase (uricase) from ox kidney powders. While the neutral and anionic detergents were found to enhance soluble urate oxidase activity, the cationic detergents, e.g., quaternary ammonium salts, were found to decrease total enzymatic activity with increasing concentration. The authors concluded that cationic detergents were not useful for purifying ox kidney urate oxidase
Solubilization of recombinant proteins, porcine growth hormone, methionyl-porcine growth hormone, infectious bursal disease virus protein, B-galactosidase fusion protein, from E. coli inclusion bodies or cells, with cationic surfactants is described in U.S. Pat. No. 4,797,474, U.S. Pat. No. 4,992,531, U.S. Pat. No. 4,966,963, and U.S. Pat. No. 5,008,377, each incorporated herein by reference in its entirety. Solubilization under alkaline conditions is accomplished using quaternary ammonium compounds including cetyltrimethylammonium chloride, mixed n-alkyl dimethyl benzylammonium chloride, CPC, N,N-dimethyl-N-[2-[2-[4-(1,1,3,3,-tetramethylbutyl)-phenoxy]ethoxy]ethyl]benzenemethanammonium chloride, tetradecyl trimethylammonium bromide, dodecyl trimethylammonium bromide, cetyl trimethylammonium bromide. These publications mention that, after each solubilization process, the solutions are centrifuged, and little to no pellet is observed in each case. This observation suggests that most or all of the proteins are solubilized without regard to selectivity for the solubilization of a target protein. The purity of the recovered proteins is not indicated. U.S. Pat. No. 5,929,231, incorporated herein by reference in its entirety, describes cetyl pyridinium chloride (CPC) disintegration of granules and aggregates containing starches. Thus, the prior art relates to use of cationic surfactants for general, nonspecific solubilization of particulate biological macromolecules. These methods of the prior art do not disclose increasing the purity of a desired target protein with respect to total protein with a cationic surfactant.
Cationic surfactants have also been used to elute biological macromolecules adsorbed to cation exchange resins or aluminum-containing adjuvants (Antonopoulos, et al. (1961) Biochim. Biophys. Acta 54:213-226; Embery (1976) J. Biol. Buccale 4:229-236; and Rinella, et al. (1998) J. Colloid Interface Sci. 197:48-56, each of which is incorporated herein by reference in its entirety). U.S. Pat. No. 4,169,764, incorporated herein by reference in its entirety, describes elution of urokinase from carboxymethyl cellulose columns using a wide variety of cationic surfactant solutions. The authors state a preference for using tetra substituted ammonium salts in which one alkyl group is a higher alkyl group up to 20 carbon atoms and the others are lower alkyl groups up to 6 carbon atoms. Use of such cationic surfactants enables removal of biological macromolecules from their attachment to a solid matrix.
Conversely, impregnation of filters such as those composed of nylon, with cationic surfactant enables immobilizing of polysaccharides or nucleic acids (Maccari and Volpi (2002) Electrophoresis 23:3270-3277; Benitz, et al. (1990) U.S. Pat. No. 4,945,086; Macfarlane (1991) U.S. Pat. No. 5,010,183, each of which is incorporated herein by reference in its entirety). This phenomenon is apparently due to cationic surfactant-polyanion interactions which enable precipitation of the polyanion.
It is well established that amphipathic ammonium compounds, which comprise quaternary ammonium compounds of the general formula QN+ and paraffin chain primary ammonium compounds of the general formula RNH3+, can precipitate polyanions under defined conditions (reviewed in Scott (1955) Biochim. Biophys. Acta 18:428-429; Scott (1960) Methods Biochem. Anal. 8:145-197; Laurent, et al., (1960) Biochim. Biophys. Acta 42:476-485; Scott (1961) Biochem. J. 81:418-424; Pearce and Mathieson (1967) Can. J. Biochemistry 45:1565-1576; Lee (1973) Fukushima J. Med. Sci. 19:33-39; Balazs, (1979) U.S. Pat. No. 4,141,973; Takemoto, et al., (1982) U.S. Pat. No. 4,312,979; Rosenberg (1981) U.S. Pat. No. 4,301,153; Takemoto, et al., (1984) U.S. Pat. No. 4,425,431; d'Hinterland, et al., (1984) U.S. Pat. No. 4,460,575; Kozma, et al. (2000) Mol. Cell. Biochem. 203:103-112, each of which is incorporated herein by reference in its entirety). This precipitation is dependent on the precipitating species having a high polyanion charge density and high molecular weight (Saito (1955) Kolloid-Z 143:66, incorporated herein by reference in its entirety). The presence of salts can interfere with or reverse cationic surfactant-induced precipitation of polyanions.
Additionally, polyanions can be differentially precipitated from solutions containing protein contaminants, under alkaline pH conditions. In such cases, proteins not chemically bound to the polyanions will remain in solution, while the polyanions and other molecules bound to the polyanions will precipitate. For example, precipitation of polyanions such as polysaccharides and nucleic acids is accompanied by co-precipitation of molecules such as proteoglycans and proteins interacting with the polyanions (Blumberg and Ogston (1958) Biochem. J. 68:183-188; Matsumura, et al., (1963) Biochim. Biophys. Acta 69: 574-576; Serafini-Fracassini, et al. (1967) Biochem. J. 105:569-575; Smith, et al. (1984) J. Biol. Chem. 259:11046-11051; Fuks and Vlodavsky (1994) U.S. Pat. No. 5,362,641; Hascall and Heinegard (1974) J. Biol. Chem. 249:4232-4241, 4242-4249, and 4250-4256; Heinegard and Hascall (1974) Arch. Biochem. Biophys. 165: 427-441; Moreno, et al. (1988) U.S. Pat. No. 4,753,796; Lee, et al. (1992) J. Cell Biol. 116: 545-557; Varelas, et al. (1995) Arch. Biochem. Biophys. 321: 21-30, each of which is incorporated herein by reference in its entirety).
The isoelectric point (or pI) of a protein is the pH at which the protein has an equal number of positive and negative charges. Under solution conditions with pH values close to (especially below) a protein's isoelectric point, proteins can form stable salts with strongly acidic polyanions such as heparin. Under conditions which promote precipitation of such polyanions, the proteins complexed with the polyanions also precipitate (L B Jaques (1943) Biochem. J. 37:189-195; A S Jones (1953) Biochim. Biophys. Acta 10:607-612; J E Scott (1955) Chem and Ind 168-169; U.S. Pat. No. 3,931,399 (Bohn, et al., 1976) and U.S. Pat. No. 4,297,344 (Schwinn, et al., 1981), each of which is incorporated herein by reference in its entirety).
U.S. Pat. No. 4,421,650, U.S. Pat. No. 5,633,227, and Smith, et al. ((1984) J. Biol. Chem. 259:11046-11051, each of which is incorporated herein by reference in its entirety) describe purification of polyanions by sequential treatment with a cationic surfactant and ammonium sulfate (that enables dissociation of polyanion-cationic surfactant complexes) and subsequent separation using hydrophobic interactions chromatography. European patent publication EP055188, incorporated herein by reference in its entirety, describes cationic surfactant-enabled separation of RTX toxin from lipopolysaccharide. However, there is no mass balance in the amount of lipopolysaccharide that is quantified by endotoxin activity assays. Neutralization of endotoxin activity by strongly interacting cationic compounds has been demonstrated (Cooper J F (1990) J Parenter Sci Technol 44:13-5, incorporated herein by reference in its entirety). Thus, in EP055188, the lack of endotoxin activity in the precipitate following treatment with increasing amounts of cationic surfactant possibly results from neutralization of the activity by surfactant-lipopolysaccharide complex formation.
The above-mentioned methods require intermediary polyanions, solid supports or aggregates comprising proteins with selective solubility by a cationic surfactant for enabling purification of soluble proteins using cationic surfactant. Hence, the prior art does not provide a method of purifying a target protein by contacting the protein with a cationic surfactant in an amount effective to preferentially precipitate proteins other than the target protein, i.e., contaminating proteins, particularly when such contacting is done in the absence of intermediary polyanions, solid supports, or aggregates of proteins. Often, one skilled in the art encounters mixtures of soluble proteins and does not have a simple, efficient means for purifying the desired protein. The novel method for purifying proteins, described herein, enables efficient purification of target proteins by using cationic surfactants to preferentially precipitate proteins other than the target protein. Preferably such precipitation of contaminating proteins is direct, and does not depend upon the presence of polyanions, solid supports or aggregates comprising the contaminating proteins and other molecules.